**Pregnancy Rates Following Transfer of Cultured Versus Non Cultured Frozen Thawed Human Embryos**

Bharat Joshi, Manish Banker, Pravin Patel, Preeti Shah and Deven Patel *Pulse Women's Hospital, Ahmedabad, Gujarat India* 

## **1. Introduction**

176 Advances in Embryo Transfer

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Veeck, L. & Zaninovic, N. (2009). Human blastocysts in vitro. In: Veeck L, Zaninovic N, eds.

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153

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mammalian preimplantation embryos: historical perspective and current issues.

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Frozen embryo transfers (FET) are increasingly becoming a routine part of *in-*Vitro Fertilization (IVF) programs at advanced assisted reproductive technology (ART) centers throughout the world. Comparatively FET yield lower pregnancy rates than fresh embryo transfers. Cryopreseravtion offers optimum utilization of embryos produced and is advantageous for the patients with the surplus embryos, ovarian hyper stimulation, radiotherapy or chemotherapy etc. It is of prime importance to dehydrate cells i.e. withdrawal of intracellular water. This is brought about by adding permeable and non permeable cryoprotective agents (CPA). These agents help to minimize the damage to the organelles within the cell and also help to maintain the osmolar changes taking place during the freezing and thawing process. Almost all the cryoprotectants exert some degree of toxic effect to the blastomeres in direct proportion to their concentration used and the time of exposure. The toxic effect can be reduced by step wise addition of cryoprotective agent during the freezing process. Freezing and thawing may damage the morphological characteristics of embryos and survival rate of blastomeres resulting into lower implantation rates. With the varying degrees of post-thaw survival (40% to 90%) and success rate (ranging from 7% to 40%) oocytes and embryos have been frozen in almost all developmental stages, e.g. oocyte (Chen,1986, Borini et al, 2004,2006), pronuclear (Barg et al.,1990, Van den Abbeel et al.,1997, Senn et al.,2000 and Al-Hasani et al.,2007), cleavage stage on day2 and day3 post OPU (Li et al.,2007, Mauri et al.,2001, Kuwayama et al.,2005, Van der Elst et al.,1997 and Rama Raju et al., 2005), morula stage (Tao et al.,2001) and blastocyst stage (Menezo,2004, Liebermann and Tucker,2006, Clifford et al.,2007).

## **2. Cryopreservation**

Cooling exerts irreversible damage to the cytoplasmic lipid droplets and reversible damage to the microtubules. Further the damages are also shown in intracellular organelles, cytoskeleton and cell-to-cell contacts (Vincent and Jhonson, 1992; Massip et al.,1995; Dobrinsky,1996). Also, due to cooling and thawing process there is fracture damage to the zona pellucida leading to the varying survival of the frozen thawed embryos. As reviewed

Pregnancy Rates Following Transfer of Cultured

**5.1 Embryo freezing and thawing at Pulse** 

using Embryo Thawing Media (Cook Medicals, Australia).

**5. Materials and methods** 

**5.2 Results and discussion** 

**Patient's AGE** 

Table 1. Pregnancy rates according to patient's age

**5.4 Number of embryos transferred** 

**No. of** 

**5.3 Patient's age** 

years (p<0.01).

Versus Non Cultured Frozen Thawed Human Embryos 179

With the aim to improve upon pregnancy rates (PR) and to get better selection of embryos from thawed embryos (culture vs non culture), a retrospective analysis of work done at Pulse Women's Hospital, Ahmedabad, India during 2006-2011 is presented here. Patients opting for ART procedure were stimulated using long or antagonist protocols. Ovulation was triggered when majority of the follicles attained 18-20mm diameter with human Chorionic Gonadotropin (hCG). Mature oocytes were inseminated within 4 hours of collection. Fertilization checks were carried out 16-20 hours post insemination. Not more than three embryos were transferred ~48 hours post egg collection. Surplus embryos were frozen in Embryo Freezing Media (Cook Medicals, Australia) using Cryologic programmable biofreezer (CL 8800) in mini straws (0.25 ml capacity). Seeding was induced at -70C followed by cooling with the rate -0.30C /minute up to -350C with a free fall to - 1200C. Thereafter the straws were stored in liquid Nitrogen. Embryos were thawed either on the ET day or a day prior to ET. Thawing was performed with stepwise removal of CPA

Data created during 2006-2011 were analyzed using Chi-Square test. Out of total 1248 frozen thaw ET cycles, 275 pregnancies (PR-22.0%) were obtained, similar to the 28.8% pregnancy rates obtained by Ying-hui et al, 2002. Results were compared for parameters like patient's age, number of embryos transferred and the time interval between thawing and transfer.

Comparing the age of patient (Table:1), up to 30 years resulted in 25.4 % pregnancy rates which is significantly higher than the 18.6% rates obtained in patients with the age of >30

≤30 630 160 25.4

>30 618 115 18.6

FET of 3 embryos yielded 27.1% pregnancy rates, significantly higher to the 9.9% pregnancy

rates (Table:2) obtained when upto 2 embryos were transferred (p<0.0003).

**ET'S PREGNANT PREGNANCY** 

**RATE (%)** 

by Guiref et al,2002 and Abdel Hafez et al,2010 various factors affecting outcome are embryo cleavage stage, pre freeze appearance, hormone supplementation during the cycle, ovarian stimulation before Ovum Pick Up (OPU), outcome of fresh Embryo Transfer (ET) cycle, choice of cryoprotective agent, , freezing technique (Controlled rate slow vs Vitrification), type of carrier etc.

## **3. Slow freezing**

Slow freezing is a process where embryos are equilibrated in 1-2 mol/l of permeable and non permeable cryoprotectants, cooled from room temperature to -7°C temperature in a controlled biological freezer, induce seeding at -7°C and than slowly cooled at 0.3 – 1.0°C/ minute up to -35°C, with a freefall up to -120°C. Embryos loaded in straws are plunged in to liquid Nitrogen for storage. Slow freezing causes damage to the embryos due to the intra cellular ice crystal formation but the toxic effect of low concentrations of cryoprotectants favours survival of embryos in slow freezing. This technique is expensive in its requirement of a programmable bio-freezer, liquid Nitrogen consumption, electricity, time of freezing etc. Still this is widely practiced freezing technique in many laboratories around the world.

## **4. Vitrification**

Vitrification is the process of solidification of a solution at low temperatures without ice crystal formation, by extreme elevation in its viscosity using cooling rates of 15,000 to 30,0000C/ min (Rama Raju et al, 2006). This phenomenon requires either rapid cooling rates (Rall,1987) or the use of high concentration of cryoprotectant solutions, which decrease ice crystal formation and increase viscosity at low temperatures until the molecules become immobilized and has the properties of solid (Fahy,1986). To achieve rapid cooling (2500°C/min), the exposure time of embryos to cryoprotectant solutions must be short due to the toxic effects of high cryoprotectant concentrations. However, if the exposure is too short, the penetration of the cryoprotectant will be inadequate and intracellular ice could form, even in the absence of extracellular ice (Otoi et al.,1998). The way to circumvent the noxious effects of cryoprotectants in the vitrification process could be through the use of high cryoprotectant concentrations for short periods of time (Vajta et al.,1998) or increasing the equilibration period by using lower cryoprotectant concentrations (Papis et al.,2000).

With the advances made in cryobiology by using combinations of freezing solution or/and thawing techniques there is an improvement in the pregnancy rates. This was extensively reviewed in a meta-analysis by Abdel Hafez et al, 2010 suggesting superiority of vitrification technology over slow freezing method in terms of significantly higher survival rate of embryos with an increased implantation and pregnancy rates. In terms of techniques employed for freezing, vitrification is gaining more and more support over slow freezing.

However, at our center vitrification has just been introduced and still in trial phase. Simultaneously we are putting in efforts to investigate factors which may enhance pregnancy rates using slow freezing with the aim to select embryos after freezing and thawing so as to get similar results as obtained by using fresh embryos.

## **5. Materials and methods**

178 Advances in Embryo Transfer

by Guiref et al,2002 and Abdel Hafez et al,2010 various factors affecting outcome are embryo cleavage stage, pre freeze appearance, hormone supplementation during the cycle, ovarian stimulation before Ovum Pick Up (OPU), outcome of fresh Embryo Transfer (ET) cycle, choice of cryoprotective agent, , freezing technique (Controlled rate slow vs

Slow freezing is a process where embryos are equilibrated in 1-2 mol/l of permeable and non permeable cryoprotectants, cooled from room temperature to -7°C temperature in a controlled biological freezer, induce seeding at -7°C and than slowly cooled at 0.3 – 1.0°C/ minute up to -35°C, with a freefall up to -120°C. Embryos loaded in straws are plunged in to liquid Nitrogen for storage. Slow freezing causes damage to the embryos due to the intra cellular ice crystal formation but the toxic effect of low concentrations of cryoprotectants favours survival of embryos in slow freezing. This technique is expensive in its requirement of a programmable bio-freezer, liquid Nitrogen consumption, electricity, time of freezing etc. Still this is widely practiced freezing technique in many

Vitrification is the process of solidification of a solution at low temperatures without ice crystal formation, by extreme elevation in its viscosity using cooling rates of 15,000 to 30,0000C/ min (Rama Raju et al, 2006). This phenomenon requires either rapid cooling rates (Rall,1987) or the use of high concentration of cryoprotectant solutions, which decrease ice crystal formation and increase viscosity at low temperatures until the molecules become immobilized and has the properties of solid (Fahy,1986). To achieve rapid cooling (2500°C/min), the exposure time of embryos to cryoprotectant solutions must be short due to the toxic effects of high cryoprotectant concentrations. However, if the exposure is too short, the penetration of the cryoprotectant will be inadequate and intracellular ice could form, even in the absence of extracellular ice (Otoi et al.,1998). The way to circumvent the noxious effects of cryoprotectants in the vitrification process could be through the use of high cryoprotectant concentrations for short periods of time (Vajta et al.,1998) or increasing the equilibration period by using lower cryoprotectant concentrations (Papis et al.,2000).

With the advances made in cryobiology by using combinations of freezing solution or/and thawing techniques there is an improvement in the pregnancy rates. This was extensively reviewed in a meta-analysis by Abdel Hafez et al, 2010 suggesting superiority of vitrification technology over slow freezing method in terms of significantly higher survival rate of embryos with an increased implantation and pregnancy rates. In terms of techniques employed for freezing, vitrification is gaining more and more support over

However, at our center vitrification has just been introduced and still in trial phase. Simultaneously we are putting in efforts to investigate factors which may enhance pregnancy rates using slow freezing with the aim to select embryos after freezing and

thawing so as to get similar results as obtained by using fresh embryos.

Vitrification), type of carrier etc.

laboratories around the world.

**4. Vitrification** 

slow freezing.

**3. Slow freezing** 

## **5.1 Embryo freezing and thawing at Pulse**

With the aim to improve upon pregnancy rates (PR) and to get better selection of embryos from thawed embryos (culture vs non culture), a retrospective analysis of work done at Pulse Women's Hospital, Ahmedabad, India during 2006-2011 is presented here. Patients opting for ART procedure were stimulated using long or antagonist protocols. Ovulation was triggered when majority of the follicles attained 18-20mm diameter with human Chorionic Gonadotropin (hCG). Mature oocytes were inseminated within 4 hours of collection. Fertilization checks were carried out 16-20 hours post insemination. Not more than three embryos were transferred ~48 hours post egg collection. Surplus embryos were frozen in Embryo Freezing Media (Cook Medicals, Australia) using Cryologic programmable biofreezer (CL 8800) in mini straws (0.25 ml capacity). Seeding was induced at -70C followed by cooling with the rate -0.30C /minute up to -350C with a free fall to - 1200C. Thereafter the straws were stored in liquid Nitrogen. Embryos were thawed either on the ET day or a day prior to ET. Thawing was performed with stepwise removal of CPA using Embryo Thawing Media (Cook Medicals, Australia).

## **5.2 Results and discussion**

Data created during 2006-2011 were analyzed using Chi-Square test. Out of total 1248 frozen thaw ET cycles, 275 pregnancies (PR-22.0%) were obtained, similar to the 28.8% pregnancy rates obtained by Ying-hui et al, 2002. Results were compared for parameters like patient's age, number of embryos transferred and the time interval between thawing and transfer.

## **5.3 Patient's age**

Comparing the age of patient (Table:1), up to 30 years resulted in 25.4 % pregnancy rates which is significantly higher than the 18.6% rates obtained in patients with the age of >30 years (p<0.01).


Table 1. Pregnancy rates according to patient's age

#### **5.4 Number of embryos transferred**

FET of 3 embryos yielded 27.1% pregnancy rates, significantly higher to the 9.9% pregnancy rates (Table:2) obtained when upto 2 embryos were transferred (p<0.0003).

Pregnancy Rates Following Transfer of Cultured

cleaved embryos were transferred.

table 4.

**EMBRYOS CLEAVED (only 8 cells)** 

**EMBRYOS** 

Versus Non Cultured Frozen Thawed Human Embryos 181

1 422 83 19.7b <0.0001

2 258 93 36.1c =0.0004

3 100 43 43d =0.0005

Table 4. Comparison of pregnancy rate between numbers of cleaved embryos transferred

When 2 cleaved embryos after culture were transferred, 36.1% pregnancies were achieved and when all 3 cultured and cleaved embryos were transferred, 43.0% pregnancy rates were achieved, showing that these results are at par with those obtained in fresh embryo transfer cycle. This is much higher than the results obtained by Karlstorm et al,1997, where pregnancy rates was 23% when 2 cleaved embryos were transferred and 27% when 3

Pregnancy rates have shown further increase when only the embryos which have cleaved to an eight cell stage during culture were transferred (Table:5) as compared to results shown in

0 294 21 7.1a

Table 5. Comparison of pregnancy rate when only 8-cell embryos were transferred

Highly significant difference (p<0.0001) in pregnancy rates was observed when all noncleaved embryos were transferred compared to 1 to 3 cleaved 8-celled embryos. Pregnancy rates were significantly higher when two (p<0.001) and three (p<0.003) cleaved 8- celled embryos were transferred compared to only one cleaved 8- celled embryo. However, there was no significant difference between transfer of two and three cleaved 8-celled embryos.

**ET PREGNANCY PREGNANCY** 

1 302 67 22.2b <0.0001 a,b

2 208 84 40.4c <0.001 b,c

3 82 38 46.3d <0.003 b,d

**RATE (%) p values** 

**RATE (%) P values** 

a.b

b,c

b,d

**CLEAVED ET PREGNANCY PREGNANCY** 

0 294 21 7.1a


Table 2. Pregnancy rates according to number of embryos transferred

## **5.5 Time interval between thawing and transfer**

Data were further divided into two groups viz. group I in which embryos were thawed and cultured overnight before ET and group II in which embryos were thawed and transferred within two hours (Table: 3).


Table 3. Pregnancy rates according to time of ET

Embryos cultured overnight produced pregnancy rates of 22.3% while embryos transferred within 2 hours post thawing resulted in pregnancy rates of 20.1%, showing no significant difference between the two groups. Results are comparable with the pregnancy rates obtained by Karlstorm et al, 1997 (22%) and by Singh et al,2007 (16%).

Van der Elst et al,1997, Zeibe et al,1998 and Guiref et al,2002 proposed to evaluate resumption of mitosis by doing overnight culture and selection of thawed embryos before transfer. On this basis within group I i.e. the cultured group, embryos were assessed for further cleavage of the blastomeres. There was a significant difference (p<0.0001) when all non-cleaved embryos were transferred (7.1%) and when 1 to 3 cleaved embryos (31.9%) were transferred (Table:4). This is similar to the results obtained by Ying-hui et al,2002, where 35.2% pregnancy rate was achieved when at least one cleaved embryo was transferred against 10.2% when no cleaved embryo was transferred. Further pregnancy rates were significantly higher when two (p=0.0004) and three (p=0.0005) cleaved embryos were transferred compared to only one cleaved embryo. However, there was no significant difference between transfer of two and three cleaved embryos.

≤ 2 365 36 9.9

3 883 239 27.1

Data were further divided into two groups viz. group I in which embryos were thawed and cultured overnight before ET and group II in which embryos were thawed and transferred

(Overnight culture) 31.6 yrs. 1074 240 22.3

(2 hours culture) 30.1yrs. 174 35 20.1

obtained by Karlstorm et al, 1997 (22%) and by Singh et al,2007 (16%).

difference between transfer of two and three cleaved embryos.

Embryos cultured overnight produced pregnancy rates of 22.3% while embryos transferred within 2 hours post thawing resulted in pregnancy rates of 20.1%, showing no significant difference between the two groups. Results are comparable with the pregnancy rates

Van der Elst et al,1997, Zeibe et al,1998 and Guiref et al,2002 proposed to evaluate resumption of mitosis by doing overnight culture and selection of thawed embryos before transfer. On this basis within group I i.e. the cultured group, embryos were assessed for further cleavage of the blastomeres. There was a significant difference (p<0.0001) when all non-cleaved embryos were transferred (7.1%) and when 1 to 3 cleaved embryos (31.9%) were transferred (Table:4). This is similar to the results obtained by Ying-hui et al,2002, where 35.2% pregnancy rate was achieved when at least one cleaved embryo was transferred against 10.2% when no cleaved embryo was transferred. Further pregnancy rates were significantly higher when two (p=0.0004) and three (p=0.0005) cleaved embryos were transferred compared to only one cleaved embryo. However, there was no significant

**pregnancy** 

**pregnancy/ ET PREGNANCY PREGNANCY** 

**PREGNANCY RATE (%)** 

**RATE (%)** 

**NO OF EMBRYOS No. of ET No. of** 

Table 2. Pregnancy rates according to number of embryos transferred

**5.5 Time interval between thawing and transfer** 

**GROUP MEAN AGE No. of** 

Table 3. Pregnancy rates according to time of ET

within two hours (Table: 3).

I

II


Table 4. Comparison of pregnancy rate between numbers of cleaved embryos transferred

When 2 cleaved embryos after culture were transferred, 36.1% pregnancies were achieved and when all 3 cultured and cleaved embryos were transferred, 43.0% pregnancy rates were achieved, showing that these results are at par with those obtained in fresh embryo transfer cycle. This is much higher than the results obtained by Karlstorm et al,1997, where pregnancy rates was 23% when 2 cleaved embryos were transferred and 27% when 3 cleaved embryos were transferred.

Pregnancy rates have shown further increase when only the embryos which have cleaved to an eight cell stage during culture were transferred (Table:5) as compared to results shown in table 4.


Table 5. Comparison of pregnancy rate when only 8-cell embryos were transferred

Highly significant difference (p<0.0001) in pregnancy rates was observed when all noncleaved embryos were transferred compared to 1 to 3 cleaved 8-celled embryos. Pregnancy rates were significantly higher when two (p<0.001) and three (p<0.003) cleaved 8- celled embryos were transferred compared to only one cleaved 8- celled embryo. However, there was no significant difference between transfer of two and three cleaved 8-celled embryos.

Pregnancy Rates Following Transfer of Cultured

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## **6. Conclusion**

Taking into account the results obtained in various categories it can be concluded that pregnancy rates can be improved through selection of better embryos and brought at par with that of fresh ET.

## **7. References**


Taking into account the results obtained in various categories it can be concluded that pregnancy rates can be improved through selection of better embryos and brought at par

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**6. Conclusion** 

**7. References** 

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**13**

*Iran* 

**Intercourse and ART Success Rates** 

*Shahid Sadoughi University of Medical Sciences, Yazd* 

*Sabzevar University of Medical Sciences, Sabzevar* 

*2Department of Obstetrics and Gynecology* 

Abbas Aflatoonian1, Sedigheh Ghandi2 and Nasim Tabibnejad1

*1Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility* 

Embryo implantation is critically dependent on a supportive uterine environment. Uterine receptivity is the culmination of a cellular and molecular transformation mediated locally by paracrine signals under the governance of ovarian steroid hormones, with cells and cytokines of the immune system playing integral roles in this process (1,2). The implantation rates and subsequent pregnancy rates in In Vitro Fertilization (IVF) programs are lower than the normal fertile population. During IVF treatment regimens, intercourse is not allowed and artificial insemination is excluded. There is a substantial body of evidence supporting the need for exposure of the female reproductive tract to semen / seminal plasma around the time of embryo implantation in order to maximize reproductive efficiency (3). The aim of this chapter is to examine the available evidences suggesting why intercourse is beneficial

In the reproductive process, seminal plasma is viewed primarily as a transport medium for spermatozoa traversing the female cervix and uterus after coitus (4, 5). However, studies in animal species show that seminal plasma also delivers to the female an array of signaling molecules that interact with epithelial cells lining the female reproductive tract. This interaction triggers local cellular and molecular changes that resemble an inflammatory response (6). In mouse and pig experiments, seminal fluid activates expression of several pro-inflammatory cytokines and chemokines in uterine epithelial cells (7-9). In turn, these factors amplify the actions of seminal fluid chemotactic agents resulting in vascular changes and recruitment and activation of macrophages, granulocytes and dendritic cells. These cells accumulate in the uterine endometrial tissue subjacent to the epithelial surface, and migrate between epithelial cells into the luminal cavity (9-11). The infiltrating leukocytes are implicated in clearance of seminal debris from the female tissues and potentially selection of fertilizing sperm (12,13). The infiltrating leukocytes may also influence the female immune response to seminal antigens and evoke tissue remodelling changes to condition the

In mice and pigs, the epithelial cells of the uterine endometrium are the primary site of seminal fluid interaction and, the induced key cytokines are GM-CSF and IL-6, as well as the chemokines KC (mouse IL-8 homologue) and MCP-1 (7-9). Experiments with male mice by vasectomy or surgical removal of the seminal vesicle gland show that the active signaling

or harmful to assisted reproductive techniques outcome.

endometrial environment in preparation for pregnancy (3,6).

**1. Introduction** 


## **Intercourse and ART Success Rates**

Abbas Aflatoonian1, Sedigheh Ghandi2 and Nasim Tabibnejad1

*1Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility Shahid Sadoughi University of Medical Sciences, Yazd 2Department of Obstetrics and Gynecology Sabzevar University of Medical Sciences, Sabzevar Iran* 

## **1. Introduction**

184 Advances in Embryo Transfer

[29] Karlstorm, P.O., Bergh, T., Forsberg, A.S. Sandkvist, U., Wikland, M. (1997) Prognostic

[30] Singh, P.M., Vrotson, K., Balen, A.H., (2007) Frozen embryo replacement cycle: An

[31] Zeibe, S., Bech, B., Petersen, K., et al., (1998) Resumption of mitosis during post-thaw

1263-1266.

240-244.

Reproduction. Vol 13, pp-178-181.

factors for the success rate of embryo freezing. Human Reproduction. Vol 12, pp-

analysis of factors influencing the outcome. J. Obstet Gynecol India. Vol 57, pp-

culture: a key parameter in selecting the right embryos for transfer. Human

Embryo implantation is critically dependent on a supportive uterine environment. Uterine receptivity is the culmination of a cellular and molecular transformation mediated locally by paracrine signals under the governance of ovarian steroid hormones, with cells and cytokines of the immune system playing integral roles in this process (1,2). The implantation rates and subsequent pregnancy rates in In Vitro Fertilization (IVF) programs are lower than the normal fertile population. During IVF treatment regimens, intercourse is not allowed and artificial insemination is excluded. There is a substantial body of evidence supporting the need for exposure of the female reproductive tract to semen / seminal plasma around the time of embryo implantation in order to maximize reproductive efficiency (3). The aim of this chapter is to examine the available evidences suggesting why intercourse is beneficial or harmful to assisted reproductive techniques outcome.

In the reproductive process, seminal plasma is viewed primarily as a transport medium for spermatozoa traversing the female cervix and uterus after coitus (4, 5). However, studies in animal species show that seminal plasma also delivers to the female an array of signaling molecules that interact with epithelial cells lining the female reproductive tract. This interaction triggers local cellular and molecular changes that resemble an inflammatory response (6). In mouse and pig experiments, seminal fluid activates expression of several pro-inflammatory cytokines and chemokines in uterine epithelial cells (7-9). In turn, these factors amplify the actions of seminal fluid chemotactic agents resulting in vascular changes and recruitment and activation of macrophages, granulocytes and dendritic cells. These cells accumulate in the uterine endometrial tissue subjacent to the epithelial surface, and migrate between epithelial cells into the luminal cavity (9-11). The infiltrating leukocytes are implicated in clearance of seminal debris from the female tissues and potentially selection of fertilizing sperm (12,13). The infiltrating leukocytes may also influence the female immune response to seminal antigens and evoke tissue remodelling changes to condition the endometrial environment in preparation for pregnancy (3,6).

In mice and pigs, the epithelial cells of the uterine endometrium are the primary site of seminal fluid interaction and, the induced key cytokines are GM-CSF and IL-6, as well as the chemokines KC (mouse IL-8 homologue) and MCP-1 (7-9). Experiments with male mice by vasectomy or surgical removal of the seminal vesicle gland show that the active signaling

Intercourse and ART Success Rates 187

and the result showed that clinical pregnancy rates were higher in patients with additional postoperative insemination (24). In one randomized, double blind study, the absence or presence of seminal fluid in patients undergoing ovulation induction with intrauterine insemination was investigated. Intercourse was restricted. A comparison of clinical pregnancy rates between two groups showed no significant difference. Further in non–participants with unregulated intercourse, the pregnancy rates were not significantly different (25). Coulam and Stern suggested that higher implantation rates were obtained in a group of women experiencing infertility and/or recurrent spontaneous abortion who received vaginal capsules of seminal plasma versus placebo, however this difference was not significant (26). The same group reported that in women experiencing a history of recurrent spontaneous abortion, seminal plasma enhanced the probability of live birth by 21% (27). Therefore the vast majority of evidence suggests that exposure of the female reproductive tract to sperm/seminal plasma through either artificial insemination or intercourse does not have negative impact on

Theoretically, intercourse can impair implantation by these mechanisms:

iii. pressure created by penile contact with the cervix may dislodge the embryos, furthermore intercourse may produce painful rupture of ovarian follicles.

Intercourse has been linked with ascending uterine infection during late pregnancy (28), and subclinical infection of the upper reproductive tract is associated with poor IVF embryo transfer outcome (29). During an IVF cycle the uterine cavity is vulnerable to intercourse related infection since the cervical mucus barrier that prevents ascending infection is disrupted by passage of the embryo transfer catheter. On the other hand, Sharkey et al investigated seminal plasma induction of inflammatory cytokines and chemokines gene regulation in human cervical vaginal epithelial cells in vitro. These experiments show that seminal plasma can elicit expression of a range of inflammatory cytokines and chemokines in reproductive epithelia, and implicate the ectocervix as the primary site of responsiveness. Seminal factor regulation of inflammatory cytokines in the cervical epithelium is implicated in controlling the immune response to seminal antigens. This inflammatory cytokines also is responsible for defense against infectious agents introduced at intercourse (30). Intercourse increases uterine myometrial activity during female orgasm (31) and these contractions may interfere implantation of early embryo since high levels of spontaneous uterine activity are associated with poor IVF outcome (32, 33). On the positive side, intercourse may act to assist implantation. Seminal plasma induction of the pro - inflammatory and chemotactic cytokines supports the interpretation because one function of seminal fluid is to activate an inflammatory cascade after deposition in the human female reproductive tract at intercourse, analogous to the consequences of mating in mice (6) and in pigs (8). Since each of these factors also regulates leukocyte recruitment and activation in humans, it is likely that elevated production of the pro - inflammatory and chemotactic cytokines in the cervix elicits changes in local leukocytes, which manifest as the post – coital leukocytic reaction in women (15, 16). Epithelial cell regulation of this response is consistent with the notion that epithelial cytokines control accumulation and functional behavior of local dendritic cell, macrophage and

outcome.

**3. Discussion** 

i. the introduction of infection,

ii. initiation of uterine contractions (at orgasm),

moieties (signaling molecules) are contained within the seminal plasma fraction of the ejaculate and are derived from the seminal vesicle (8). Cytokines of the transforming growth factor-β (TGF-Β) family are identified as the major active factors in mouse seminal plasma (14). Seminal TGF-B is synthesized in the latent form in the seminal vesicle gland and is activated in the female tract upon deposition at mating (14).

However, how seminal fluid activates inflammatory cytokine synthesis or leukocyte infiltration in any compartment of the human female tract is unclear. Two previous *in vivo* studies in women have shown neutrophil exocytosis in the cervical tissues following either sexual intercourse or artificial insemination and they reported that sperm, but not seminal plasma, is required to elicit this "leukocytic reaction" (15,16). There are preliminary indications that signaling activity is also associated with the cell-free, plasma fraction of semen. *In vitro* studies demonstrate that human female reproductive tract cells can respond to seminal plasma, with increased IL-8 and secretory leukocyte protease inhibitor (SLPI) secretion from cervical explants (17). Endometrial epithelial cells are reported to show elevated synthesis of IL-1β, IL-6 and leukaemia inhibitory factor (LIF) after culture with seminal plasma (18). Seminal fluid might also target infiltrating leukocytes directly, since IL-10 production in human monocyte U937 cells can be induced in response to seminal fluid constituents (17).Transmission of seminal factors in the female reproductive tract organizes molecular and cellular changes in the endometrium to facilitate embryo development and implantation.

## **2. Clinical studies**

The available literature on the potential benefit of intercourse in patients undergoing assisted reproduction techniques is not extensive. To date; only two studies have examined the effect of intercourse around the time of embryo transfer on ART cycles (19, 20), while several studies have looked at the effect of artificial insemination with whole semen or seminal plasma (21- 23). In a multicenter randomized study, patients undergoing fresh (400 cycles) and thawed (200 cycles) embryo transfer were randomized either to abstain or to engage in vaginal intercourse around the time of embryo transfer. There were no significant difference in pregnancy rates between the intercourse and abstinence groups but viability of transferred embryos at 6 – 8 weeks was significantly higher in the exposed semen group than abstained group. The authors' result indicates that exposure to semen around the time of embryo transfer increases the likelihood of successful early embryo implantation and development (19). In another study, 390 women were randomly divided into intercourse and abstinence groups during embryo transfer period and results indicated that intercourse did not significantly increase the pregnancy and implantation rates in ART cycles (20). Clinical pregnancy rates were not significantly different in two groups. In a prospective trial, Bellinge demonstrated an improved implantation rate with high vaginal deposition of a portion of their partners' semen samples compared with controls (21). However, in a subsequent prospective trial, Fishel found no significant effect of the use of high vaginal insemination at the time of oocyte recovery in patients undergoing IVF (22). Neither of these two reports utilized true randomization methods to assign treatment – control protocols. The final randomized control trial allocated patients to high vaginal insemination with either their partners' seminal plasma or a saline placebo at the time of oocyte retrieval (23). This study of 168 patients reported a 45% relative increase in implantation rates in the seminal plasma exposed group, but it did not reach statistical significance. In one retrospective study, postoperative intrauterine and intracervical insemination was performed in gamete intrafallopian transfer (GIFT) program

moieties (signaling molecules) are contained within the seminal plasma fraction of the ejaculate and are derived from the seminal vesicle (8). Cytokines of the transforming growth factor-β (TGF-Β) family are identified as the major active factors in mouse seminal plasma (14). Seminal TGF-B is synthesized in the latent form in the seminal vesicle gland and is

However, how seminal fluid activates inflammatory cytokine synthesis or leukocyte infiltration in any compartment of the human female tract is unclear. Two previous *in vivo* studies in women have shown neutrophil exocytosis in the cervical tissues following either sexual intercourse or artificial insemination and they reported that sperm, but not seminal plasma, is required to elicit this "leukocytic reaction" (15,16). There are preliminary indications that signaling activity is also associated with the cell-free, plasma fraction of semen. *In vitro* studies demonstrate that human female reproductive tract cells can respond to seminal plasma, with increased IL-8 and secretory leukocyte protease inhibitor (SLPI) secretion from cervical explants (17). Endometrial epithelial cells are reported to show elevated synthesis of IL-1β, IL-6 and leukaemia inhibitory factor (LIF) after culture with seminal plasma (18). Seminal fluid might also target infiltrating leukocytes directly, since IL-10 production in human monocyte U937 cells can be induced in response to seminal fluid constituents (17).Transmission of seminal factors in the female reproductive tract organizes molecular and cellular changes in the endometrium to facilitate embryo development and implantation.

The available literature on the potential benefit of intercourse in patients undergoing assisted reproduction techniques is not extensive. To date; only two studies have examined the effect of intercourse around the time of embryo transfer on ART cycles (19, 20), while several studies have looked at the effect of artificial insemination with whole semen or seminal plasma (21- 23). In a multicenter randomized study, patients undergoing fresh (400 cycles) and thawed (200 cycles) embryo transfer were randomized either to abstain or to engage in vaginal intercourse around the time of embryo transfer. There were no significant difference in pregnancy rates between the intercourse and abstinence groups but viability of transferred embryos at 6 – 8 weeks was significantly higher in the exposed semen group than abstained group. The authors' result indicates that exposure to semen around the time of embryo transfer increases the likelihood of successful early embryo implantation and development (19). In another study, 390 women were randomly divided into intercourse and abstinence groups during embryo transfer period and results indicated that intercourse did not significantly increase the pregnancy and implantation rates in ART cycles (20). Clinical pregnancy rates were not significantly different in two groups. In a prospective trial, Bellinge demonstrated an improved implantation rate with high vaginal deposition of a portion of their partners' semen samples compared with controls (21). However, in a subsequent prospective trial, Fishel found no significant effect of the use of high vaginal insemination at the time of oocyte recovery in patients undergoing IVF (22). Neither of these two reports utilized true randomization methods to assign treatment – control protocols. The final randomized control trial allocated patients to high vaginal insemination with either their partners' seminal plasma or a saline placebo at the time of oocyte retrieval (23). This study of 168 patients reported a 45% relative increase in implantation rates in the seminal plasma exposed group, but it did not reach statistical significance. In one retrospective study, postoperative intrauterine and intracervical insemination was performed in gamete intrafallopian transfer (GIFT) program

activated in the female tract upon deposition at mating (14).

**2. Clinical studies** 

and the result showed that clinical pregnancy rates were higher in patients with additional postoperative insemination (24). In one randomized, double blind study, the absence or presence of seminal fluid in patients undergoing ovulation induction with intrauterine insemination was investigated. Intercourse was restricted. A comparison of clinical pregnancy rates between two groups showed no significant difference. Further in non–participants with unregulated intercourse, the pregnancy rates were not significantly different (25). Coulam and Stern suggested that higher implantation rates were obtained in a group of women experiencing infertility and/or recurrent spontaneous abortion who received vaginal capsules of seminal plasma versus placebo, however this difference was not significant (26). The same group reported that in women experiencing a history of recurrent spontaneous abortion, seminal plasma enhanced the probability of live birth by 21% (27). Therefore the vast majority of evidence suggests that exposure of the female reproductive tract to sperm/seminal plasma through either artificial insemination or intercourse does not have negative impact on outcome.

## **3. Discussion**

Theoretically, intercourse can impair implantation by these mechanisms:


Intercourse has been linked with ascending uterine infection during late pregnancy (28), and subclinical infection of the upper reproductive tract is associated with poor IVF embryo transfer outcome (29). During an IVF cycle the uterine cavity is vulnerable to intercourse related infection since the cervical mucus barrier that prevents ascending infection is disrupted by passage of the embryo transfer catheter. On the other hand, Sharkey et al investigated seminal plasma induction of inflammatory cytokines and chemokines gene regulation in human cervical vaginal epithelial cells in vitro. These experiments show that seminal plasma can elicit expression of a range of inflammatory cytokines and chemokines in reproductive epithelia, and implicate the ectocervix as the primary site of responsiveness. Seminal factor regulation of inflammatory cytokines in the cervical epithelium is implicated in controlling the immune response to seminal antigens. This inflammatory cytokines also is responsible for defense against infectious agents introduced at intercourse (30). Intercourse increases uterine myometrial activity during female orgasm (31) and these contractions may interfere implantation of early embryo since high levels of spontaneous uterine activity are associated with poor IVF outcome (32, 33). On the positive side, intercourse may act to assist implantation. Seminal plasma induction of the pro - inflammatory and chemotactic cytokines supports the interpretation because one function of seminal fluid is to activate an inflammatory cascade after deposition in the human female reproductive tract at intercourse, analogous to the consequences of mating in mice (6) and in pigs (8). Since each of these factors also regulates leukocyte recruitment and activation in humans, it is likely that elevated production of the pro - inflammatory and chemotactic cytokines in the cervix elicits changes in local leukocytes, which manifest as the post – coital leukocytic reaction in women (15, 16). Epithelial cell regulation of this response is consistent with the notion that epithelial cytokines control accumulation and functional behavior of local dendritic cell, macrophage and

Intercourse and ART Success Rates 189

[5] Aumuller G, Riva A. Morphology and functions of the human seminal vesicle. Andrologia

[6] Robertson SA. Seminal plasma and male factor signalling in the female reproductive tract.

[7] Robertson SA, Mayrhofer G, Seamark RF. Uterine epithelial cells synthesize granulocyte-

[8] Robertson SA, Mau VJ, Tremellen KP, et al. Role of high molecular weight seminal vesicle

[9] O'Leary S, Jasper MJ, Warnes GM, et al. Seminal plasma regulates endometrial cytokine

[10] Phillips DM, Mahler S. Migration of leukocytes and phagocytosis in rabbit vagina. J Cell

[11] McMaster MT, Newton RC, Dey SK, et al. Activation and distribution of inflammatory

[12] Mattner PE. Phagocytosis of spermatozoa by leucocytes in bovine cervical mucus in vitro.

[13] Roldan ER, Gomendio M, Vitullo AD. The evolution of eutherian spermatozoa

[14] Tremellen KP, Seamark RF, Robertson SA. Seminal transforming growth factor beta1

[16] Thompson LA, Barratt CL, Bolton AE, et al. The leukocytic reaction of the human uterine

[17] Denison FC, Grant VE, Calder AA, et al. Seminal plasma components stimulate interleukin-8 and interleukin-10 release. Mol Hum Reprod 1999;5:220-226. [18] Gutsche S, von Wolff M, Strowitzki T, et al. Seminal plasma induces mRNA expression of

[19] Tremellen KP, Valbuena D, Landeras J, Ballesteros A, Martinez J, Mendoza S,et all. The

[20] Aflatoonian A., Ghandi S., Tabibnejad N. The effect of entercourse around Embryo

[21] Bellinge BS, Copeland CM , Thomas TD, et al. The influence of patient insemination on

[22] Fishel S, Webster J, Jackson P, Faratian B. Evaluating of high vaginal insemination at

[23] Von Wolff M, Rosner S, Thone C, et al. Intravaginal and intracervical application of

macrophage colony-stimulating factor and interleukin-6 in pregnant and

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granulocyte populations in other epithelia (34). However, since the effects of seminal fluid on leukocytes have not examined, the possibility of direct seminal fluid signaling in these cells contributing to the response, cannot be excluded. Active synthesis of a wider range of cytokines in the cervix after intercourse would facilitate optimal competence in protecting the higher reproductive tract from pathogen invasion and in reinforcing the defensive barrier function of this epithelial surface (35).

Induction of GM-CSF and IL-6 in the cervical tissues would influence the activation status of local antigen-presenting cells, programming phenotypes that impact the ensuing response to antigens processed by those cells (36-39). The significance of these two cytokines being preferentially expressed in the ectocervix is consistent with this tissue being the primary site for female 'sampling' of paternal antigens. Their presence together with the action of the immune-deviating agents TGFβ and prostaglandin E2 (PGE2) in seminal fluid would be expected to ensure that the outcome of any antigen-specific immune response did not adversely affect female tolerance of any future exposure to semen. Similarly, uptaking and processing of male antigens in semen may provide an opportunity for priming the maternal immune response in preparation for an ensuing pregnancy fathered by the same male, since the conceptus shares many of the same paternal antigens (13, 39). IL-6, together with LIF and IL-1β, can also be induced in uterine endometrial cells by seminal factors *in vitro* (18), where it appears to be a key determinant of uterine receptivity at embryo implantation (40,41). This suggests that the action of seminal fluid in regulating the quality of female tract immune responses may extend higher into the female tract, after transport of active seminal constituents by uterine peristaltic contractions which transport macromolecular material as high as the fallopian tube (42). Whether GM-CSF, IL-8 and IL-6 can also be induced in endometrial cells or not needs to be examined.

## **4. Conclusion and recommendations**

A large of randomized control trials suggest that intercourse around the time of embryo transfer improve IVF implantation rates and increase pregnancy rates in ART cycle, but some studies thought that intercourse did not significantly increase pregnancy rate. While this mechanism of intercourse improving pregnancy rate is not fully understood, it seems that semen/seminal plasma could induce immune reactions in female reproductive tract that augments embryo development and endometrial receptivity. On the negative side, hyperstimulated ovaries are vulnerable to rupture during intercourse. Therefore, we suggest that intercourse around the time of embryo transfer should be encouraged, except in the small subgroup of women with large hyperstimulated ovaries.

## **5. References**


granulocyte populations in other epithelia (34). However, since the effects of seminal fluid on leukocytes have not examined, the possibility of direct seminal fluid signaling in these cells contributing to the response, cannot be excluded. Active synthesis of a wider range of cytokines in the cervix after intercourse would facilitate optimal competence in protecting the higher reproductive tract from pathogen invasion and in reinforcing the defensive barrier

Induction of GM-CSF and IL-6 in the cervical tissues would influence the activation status of local antigen-presenting cells, programming phenotypes that impact the ensuing response to antigens processed by those cells (36-39). The significance of these two cytokines being preferentially expressed in the ectocervix is consistent with this tissue being the primary site for female 'sampling' of paternal antigens. Their presence together with the action of the immune-deviating agents TGFβ and prostaglandin E2 (PGE2) in seminal fluid would be expected to ensure that the outcome of any antigen-specific immune response did not adversely affect female tolerance of any future exposure to semen. Similarly, uptaking and processing of male antigens in semen may provide an opportunity for priming the maternal immune response in preparation for an ensuing pregnancy fathered by the same male, since the conceptus shares many of the same paternal antigens (13, 39). IL-6, together with LIF and IL-1β, can also be induced in uterine endometrial cells by seminal factors *in vitro* (18), where it appears to be a key determinant of uterine receptivity at embryo implantation (40,41). This suggests that the action of seminal fluid in regulating the quality of female tract immune responses may extend higher into the female tract, after transport of active seminal constituents by uterine peristaltic contractions which transport macromolecular material as high as the fallopian tube (42). Whether GM-CSF, IL-8 and IL-6 can also be induced in

A large of randomized control trials suggest that intercourse around the time of embryo transfer improve IVF implantation rates and increase pregnancy rates in ART cycle, but some studies thought that intercourse did not significantly increase pregnancy rate. While this mechanism of intercourse improving pregnancy rate is not fully understood, it seems that semen/seminal plasma could induce immune reactions in female reproductive tract that augments embryo development and endometrial receptivity. On the negative side, hyperstimulated ovaries are vulnerable to rupture during intercourse. Therefore, we suggest that intercourse around the time of embryo transfer should be encouraged, except in the

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Yoshinaga K , editors . Blastocyst implantation. Norwell , MA: Serono Symposia

function of this epithelial surface (35).

endometrial cells or not needs to be examined.

**4. Conclusion and recommendations** 

Press. 1989; 231 – 280

Sons, Inc., New York; 1964

**5. References** 

small subgroup of women with large hyperstimulated ovaries.


**Part 5** 

**Embryo Implantation and Cryopreservation** 

cycles – a double blind , placebo – controlled , randomized pilot study . Fertil Steril 2009 ; 91:167 – 72

