Higher Pregnancy rates with a simple method for Fallopian Tube Sperm Perfusion, using the Cervical Clamp Double Nut Bivalve Speculum in the treatment of unexplained infertility : A prospective randomised study.

 Leon Mamas

 “Mitera” Maternity and Surgical Hospital,   6 Er. Stavrou, St.,  Marousi  151 23,  Athens,  Greece.

 Correspondence should be addressed at:  8 Ethn.  Makariou  Street  154 52  P. Psichico,  Athens,  Greece 

Published in Human Reproduction Journal (www.humrep.oxfordjournals.org)

 The object of this study was to evaluate the efficacy of the newly developed cervical clamp Double Nut Speculum used for Fallopian tube Sperm Perfusion (FSP) with 4 ml of the inseminate, in comparison with standard Intrauterine Insemination (IUI) using a volume of  0,5 ml of the inseminate. A hundred and four (104) couples with unexplained infertility, undergoing 202 cycles,  were enrolled in this study, assigned randomly to either IUI (group A, n=92) or FSP + DNB Speculum^® (group B, n=110). Controlled ovarian stimulation was achieved by using three different ovarian stimulation protocols in both groups. The  age of the patients  was almost similar in both groups,  as well as the follicular development. The serum hormonal measurements and the endometrial thickness was also similar on the day of  human chorionic gonadotrophin (HCG) administration. The mean (± SD) number of motile spermatozoa inseminated was 44.83 ± 16.57 ×  10⁶  in group A and 42.68 ±  13.44 × 10⁶ in group B. In group A (IUI), 11 clinical pregnancies  (presence of gestational  sac with heart beats) occurred  (11.95% per cycle ). In group B (FSP + DNB Speculum^®) 29 clinical pregnancies occurred (26.36% per cycle). These differences were statistically significant (P<0.001). The results of this study for the treatment of unexplained infertility indicate that this simple, well tolerated, inexpensive method of using the cervical clamp Double Nut  Bivalve Speculum (DNB Speculum^®) for fallopian tube sperm perfusion (FSP) is more successful than standard intrauterine insemination (IUI).

 Key  words: Cervical clamp Double Nut Bivalve Speculum (DNB Speculum^®) / Fallopian tube Sperm Perfusion (FSP) / Intrauterine Insemination (IUI) / ovarian stimulation / unexplained infertility.

 Introduction

Intrauterine insemination with mild ovarian stimulation  has in recent years been a popular method with a great interest in the treatment of unexplained infertility. The fecundity reported in the literature ranged from 5,7% to 17,7% per cycle (Allen et al., 1985; Chaffkin et al., 1991; Dodson and Hanney, 1991; Balasch et al., 1994; Ombelet et al., 1995 Campana et al., 1996). Although the number of available oocytes increase by the ovarian stimulation to 3-4 or even more, the results are still not very promising, mainly because of low sperm count,  suboptimal spermatozoa or total absence of spermatozoa at the site of fertilization.   Mortimer and Templeton  (1982) showed that there is a reduction in sperm number of five to six orders of magnitude along the length of female reproductive tract. Even, when all the characteristics of semen were normal, there was still only a 49% chance of spermatozoa being found in the peritoneal cavity. However, significantly greater numbers of peritoneal spermatozoa have been found after artificial insemination into the external os, rather than after intercourse.  Another study (Ripps et al., 1994) showed that the number of spermatozoa recovered in the peritoneal fluid at laparoscopy after IUI was very low, however the number was increasing  after utero-tubal flushes. Utero-tubal flushes required a certain intrauterine perfusion pressure for the achievement of spill  in normal tubes or in tubes with minimal adhesions (Baker and Adamson. 1995).  In the mid follicular phase and before ovulation the endometrial glandural lumen of the fallopian tube is narrow and contains some quantities of granular and membranous material, as well as  increasing amounts of cytoplasmic debris and cellular fragments ( Amso et al., 1994). A partial tubal  luminal obstruction may also be due to the presence of tubal ostium membranes (Coeman et al., 1995).

Various methods for increasing the number of spermatozoa in the  fallopian tubes at the time of ovulation were invented and tried with encouraging results. However,  these are invasive, laparoscopic (Berger, 1987) or less invasive transcervical  methods with the possibility of adverse effects, namely trauma, infection, and vasovagal episodes. For these reasons the relative benefits of the above mentioned approaches have been questioned (Lucena et al., 1989;  Pratt et al., 1991,  Oei et al.,1992).

Recently, other non invasive methods were introduced, simpler, not requiring catheterization. These methods were based on a certain injection pressure needed in order to enable the 4 ml of sperm suspension, after filling the uterine cavity with the volume of 0.4ml (Franco et al., 1992), to pass through the interstitial part of the tube and, finally, reach the ampulla and the Pouch of Douglas (Gleicher et al., 1992). These methods require an appropriate cervical seal which will prevent sperm leakage into the vagina during uterotubal perfusion, which otherwise occurs mainly because of the opening of the cervical canal due to ovulation.

Kahn et al., 1992, 1993a, 1993b, described a method of fallopian  tube sperm perfusion using an Allis clamp placed on the cervix. However, this clamping of the cervix produced some discomfort or even pain and occasionally bleeding from the cervix. Another method was described by Li, T.C., 1993. This author used a Foley  size 8 pediatric catheter with the balloon inflated in the uterine cavity to seal the uterocervical junction. However, introducing the Foley catheter through the cervix presented some difficulties, mainly because of its flexibility, especially in nulliparous patients who constitute the majority. The use of forceps was necessary to provide steady downward traction on the cervix which served to reduce the angle between the uterine cavity and the cervical canal. The discomfort and the possible endometrial injury caused by the compressive effect of the balloon are some of the disadvantages of this method.

More recently, another method for Fallopian tube sperm perfusion has been proposed (Fanchin et al., 1995a) using the Fallopian Sperm Transfer System (FAST System ^®, CCD Laboratories, Paris, France) with pregnancy results twice as high as standard IUI, but in some cases sperm reflux was noticed, due to a deficient adjustment of the acorne tip of the devise to the external cervical os. The presence of another person was also necessary  to assist in handling the syringe adapted to the vacuum valve.

To surmount the main failings of these methods, a speculum with specially modified tips was designed to fit around the cervix, allowing also easy grasping of the cervix and firm clamping of the cervical canal for the prevention of  leakage or reflux  during and after the intrauterine insemination for fallopian tube sperm perfusion. The use of this cervical clamp Double Nut Bivalve Speculum  (DNB Speculum^®, Parthenon Medical Ltd, Athens, Greece) results in a most satisfactory higher sperm perfusion. It is a simple method, non invasive, well tolerated, without any discomfort or bleeding and quite inexpensive (Mamas, L., 1995a, 1995b). A prospective randomized trial comparing results of Fallopian tube Sperm Perfusion using the DNB speculum ^®, versus standard IUI is reported here.

 Materials and methods

 Patient  selection

During the 8-month period from October 1, 1995, to May 31, 1996 a hundred and four (104) couples  undergoing 202 cycles, were referred  to the Sperm Laboratory of the hospital. They were asked whether they would like to participate in this study. All couples suffered from unexplained infertility and had regular  menstrual cycles 25-33 days. Spontaneous ovulation was checked by vaginal ultrasound and normal serum progesterone concentrations by midluteal phase serum (P > 10 ng/ml). Serum levels of follicle stimulating hormone, luteinizing hormone, prolactin, testosterone, sex hormone-binding globulin had to be in the normal range. The mean duration of infertility was 3.94 ± 1.25 years (2-8) for group A, and 3.90 ± 1.24 years (2-8) for group B. Patients had to be younger than 39 years old.

All patients had undergone hysterosalpingogram or hysterosalpingogram, laparoscopy  and hysteroscopy in the last 12 months before insemination and had had normal uterine cavity and patent tubes.

Patients  should have  normal sperm parameters (WHO, 1992) Volume: 2.0 ml or more, concentration: 20 × 10⁶  spermatozoa per ml or more, motility: 50% motile spermatozoa, forward progression (categories ‘a’ and ‘b’) or 25% or more with rapid progression (category ‘a’), morfology: 30% or more with normal forms.

Controlled ovarian stimulation protocols

Three different protocols were used for controlled ovarian stimulation (Fanchin et al., 1995b, Schlaff et al., 1995): a) clomiphene citrate + HMG, b) FSH + HMG, c) GnRH-a +  FSH + HMG. The choice of the protocol was determined by individual preferences of the physicians in our ovulation induction program.

Clomiphene Citrate (CC) and Human Menopausal Gonadotrophin  (n = 97)

This ovarian stimulation protocol consisting of clomiphene citrate 100 mg daily from day 3 to 7 of the cycle, and 75 IU daily of human menopausal gonadotrophin (HMG, Humegon FD^®; Organon, Oss, The Netherlands) from day 6-9 of the cycle. For some of the women, the administration of HMG was continued. In others it was increased to 150 IU, depending on the ovarian response. The ovarian response was monitored from day 10 of the cycle by vaginal ultrasound measurements of the number and diameter of follicles, the thickness and the morphology  of the endometrium, and by daily measurement of serum oestradiol (E₂). Human chorionic gonadotropin (HCG, Pregnyl^®; Organon) 5000-10000 IU was administered when the leading follicle was 18-19 mm in diameter and 2-3 follicles of 16-17 mm were also present (serum E₂ levels per follicle 18-19 mm of diameter; E₂> 250pg/ml). The treatment cycles were canceled  when serum oestradiol concentration exceeded 1500pg/ml or more and  six follicles or more had matured.

Follicle Stimulating Hormone and Human Menopausal Gonadotrophin  (n=88)    

This protocol started with two ampoules of purified preparation of FSH ( 75IU Metrodin^®; Serono Laboratories) that were administered  im on cycle days 2 and 4. Ovarian stimulation was then continued with one ampoule HMG (75 IU Pergonal ^® Serono Laboratories ) daily from day 6 of the cycle until the day 5000-10000 IU HCG (HCG, Profasi^®; Serono Laboratories)  was administered, when follicular maturity was achieved (leading follicle 18-19 mm in diameter, serum E₂ levels per follicle 18-19 mm of diameter; E₂> 250pg/ml).

GnRH Agonist (GnRH-α) and FSH and HMG (n=17)

 This short protocol of GnRH-α with gonadotrophin started from cycle day 2 with daily administration of one ampoule s.c. of a GnRH-α (0.1 mg triptoreline Arvekap^®; Ipsen /Biotech Laboratories, Paris, France) and continued until 10000 IU HCG (HCG, Pregnyl^®; Organon) was injected. From cycle day 4 and for four days two ampoules FSH (75 IU Metrodin^®; Serono Laboratories) were given. From cycle day 8, ovarian stimulation was continued with two ampoules HMG (75 IU Humegon FD^®; Organon) with further individual adjustment, depending on the ovarian response until HCG injection. The follicular maturation criteria  remained the same as the two preceding protocols.

Randomization

The women for treatment with either IUI or FSP + DNB Speculum ^® were chosen at random on the day of HCG administration according to their serial number. Those with odd numbers were allocated to IUI treatment, whereas women with even numbers were allocated to FSP + DNB Spec. treatment.

Sperm preparation 

     Sexual intercourse was prohibited 2 days before (HCG) administration. The semen was collected by masturbation 2 to 3 hours before insemination. The liquefied samples were analysed using a Makler counting chamber according  to World Health Organization  guidelines (WHO, 1992).

 For sperm preparation a discontinuous two-layer Percoll gradient was used. A layer of 2.5 ml of 40% Percoll  (Pharmacia Biotech AB, Upsalla, Sweden)  was layered over 2.5 ml of 80% Percoll in a 15-ml conic centrifuge tube. The undiluted semen was layered over this gradient, and the tube was centrifuged for 20 minutes at 300 × g. The fluffy mass of sperm at the bottom of the 80% Percoll layer was collected by a Pasteur pipette, resuspended  in 2 ml of Earle’s balanced salt solution (EBSS Gibgo BRL, Life Technologies, Paisley, Scotland) with 2% HSA, and centrifugation was repeated  at 200 × g for 5 minutes. The supernatant then was discarded  and  the final pellet was resuspended either in 0.5ml EBSS solution with 2% HSA for standard IUI or in 4 ml for Fallopian tube Sperm Perfusion. Both solutions were also  analysed. 

Figure 1: A. Diagram showing the DNB Speculum^®, closed, clamping the cervical canal, with the modified tips (B - C) enveloping the cervix, while the insemination catheter is in  the uterine cavity and the sperm suspension inseminated and perfused in the fallopian tube

Insemination was in both groups performed 34 - 37 hours after HCG administration. Standard IUI was performed using a common vaginal speculum. The cervix and vaginal fornices were rinsed with normal saline solution and IUI was performed using  a 1ml  plastic syringe which was filled with 0.5 ml inseminate. The syringe was attached to an 18 mm insemination catheter (Wallace, UK) which was introduced into the upper part of the uterine cavity.

Fallopian tube sperm perfusion was performed using the cervical clamp Double Nut Bivalve Speculum (DNB Speculum^® - Figure 1,A) which was specially  designed for this purpose. This cervical clamp  speculun is made of non-toxic plastic, for one use only. It comprises of two rigid blades which end in slightly squared tips  with rounded corners. These modified tips (Figure 1,B and C) easily grasp and clamp the cervix exerting pressure on the cervical canal. In this way the possibility of any leakage or reflux of the inseminate is prevented. The tips of the blades, which reach both the anterior and the posterior vaginal vault, are opened with the help of the first screw-nut, allowing good visibility of the cervix. At this point, with the speculum in the opening position, the 18 mm insemination catheter (Wallace, UK) is atraumatically  inserted through the cervical canal into the upper part of the uterine cavity, whereas the second screw-nut is close to the posterior blade handle. Then, the first screw-nut is released and the speculum is closed by the pressure of the vaginal vault. At the same time the second nut is also released and, by pushing the handle of the anterior blade, the specially modified tips of the speculum  close around the cervix and lock. A slight pressure is exerted on the clamped cervix  by the enveloping, modified tips of the speculum, not at all inconvenient to the patient. At this stage exactly, with the cervix securely clamped, insemination of 4ml of the capacitated sperm is performed  slowly,  2ml/min and no leaking is usually observed. The tubal  ostia open up by the pressure in the uterine cavity as it gradually builds up,  allowing the sperm suspension medium enter the Fallopian tubes. In this way, the number of spermatozoa is high (sperm perfusion ) throughout all the genital  tract   (uterine cavity, ampulla, Pouch of Douglas ) and especially at the sites where fertilization of the ovum takes place. The speculum is kept locked, clamping the cervix for five minutes at least.

Luteal  support

The luteal phase was supported  either by i.m. injections of 5000 IU HCG on days 2 and 7 after sperm insemination or   by oral supplementation with 300 mg micronized progesterone (Utrogestan^® Laboratories Besins Incovesco, Paris, France)    

Results

      On the day of randomisation (day of HCG administration)  ovarian stimulation protocols (CC and HMG; FSH and HMG; GnRH-α, FSH and HMG) appeared almost homogenous in both groups (Table I). There was no difference between the two groups regarding the number of mature follicles (> 17 mm of diameter ), serum E₂ levels, and the thickness of endometrium as measured by vaginal ultrasound. The total number of highly motile (graded a,b) spermatozoa, obtained after Percoll gradients, was also equivalent in both groups 44.83± 16.57 × 10⁶ for group A and 42.68± 13.44 × 10⁶ for group B.

Clinical pregnancy was defined by the presence of fetal heart beat, detected by ultrasound examination. There were 11 pregnancies in group A (IUI) 11.95% per cycle and 21.15% per  patient in four cycles, and 29 pregnancies in group B (FSP + DNB Speculum^®) 26.36% per cycle and 55.76% per patient in four cycles. The differences between the two groups treated, both in pregnancy rate per cycle and pregnancy rate per patient, were statistically significant, P<0.001. One missed abortion occurred among the patients in group A (IUI) and one twin pregnancy.  Three missed abortions occurred among the patients in group B (FSP + DNB Spec) as well as three twin pregnancies and two sets of quadruplets, which were reduced to twin pregnancies. Six cases of mild ovarian hyperstimulation syndrome (OHSS) occurred in both groups (Schenker, 1993). No case of moderate or severe OHSS was observed in both groups.

^b Total  number of sperm with forward  progressive motility (‘a’ and ‘b’)

 Discussion

 The purpose of this prospective, randomized study was to indicate the significant improvement of pregnancy rates in couples with unexplained infertility when  treated with fallopian tube sperm perfusion  (inseminate  4 ml)  by using the new DNB Speculum^® for cervical clamp, in comparison to standard IUI (inseminate  0,5). Pregnancy rates 26.36% versus 11.95% respectively. The same protocols for controlled ovarian stimulation was used in both groups. There was not a statistically significant difference  regarding the age of the patients treated, the mean number of follicles, serum E_2, endometrial thickness on the day of HCG administration and the total number of motile spermatozoa inseminated.

In previous studies the pregnancy rate per cycle in couples with unexplained infertility after controlled ovarian stimulation when FSP with  4 ml of inseminate was achieved by the use of different methods, has been reported to be in the range 24.1% - 40.0 % (Kahn et al., 1992, 1993a, 1993b, Li, T.C. 1993, Fanchin et al. 1995a). The results reported in this study concerning the FSP group on which the DNB Speculum^® was used are similar. The pregnancy rate per cycle in cases with unexplained  infertility when standard IUI was performed using a volume of  0.5 ml of inseminate has also been reported to be in the range of 5.7 - 17.7% (Evans et al., 1991; Martinez et al., 1991; Dodson and Haney,1991; Ombelet et al., 1995; Campana et al., 1996). This is comparable to the pregnancy rate of 11,95% obtained with standard IUI in the present study.

 Table II. The distribution of pregnancy rates per cycle after three cycles of treatment (190 cycles)

Studies on the dynamics of sperm transport have shown that there is a progressive decline in the numbers of spermatozoa along the length of the female tract, so that in normal Fallopian tubes a maximum of only 200 spermatozoa are present in the ampulla, although this number is increased to several hundreds or even thousands (maximum recorded 23000) in hydrosalpinges (Mortimer, D. 1983). Significantly greater numbers of peritoneal spermatozoa have been found after artificial insemination into the external os rather than after intercourse (Mortimer and Templeton, 1982). The number of spermatozoa recovered in the peritoneal fluid at laparoscopy after IUI was still very low, ranged from 2053 to 29450.  However, the number of spermatozoa recovered in the peritoneal fluid after uterotubal flushes was  significantly greater and ranged from 3500 to 9 ´ 10⁶ (Ripps et al., 1994).

Taking under serious consideration these observations, the cervical clamp Double Nut Bivalve Speculum was developed to support the method of  insemination because it achieves easily and comfortably for the patient, better fallopian tube perfusion which results in higher pregnancy rates.  

These better results may be attributed to certain well observed and reported facts:

1. To the presence of higher concentration of motile spermatozoa around the oocytes at the site of fertilization, as well as the Pouch of  Douglas.

2.    To the intrauterine insemination pressure of 70-200mmHg, necessary for the achievement of perfusion and spill in normal tubes or tubes with minimal adhesions (Baker and Adamson 1995). This pressure is also necessary because in mid-follicular phase and before ovulation the endometrial glandular lumen of the fallopian tube is narrowed by some quantities of granural and membranous material (Amso et al., 1994) and by a partial tubal luminal obstruction due to the presence of tubal ostium membranes (Coemanet al., 1995).

3.To the cervical clamping, necessary to prevent the possibility of any leaking. Observations made during insemination showed that a higher volume than 0,4ml of inseminate, which fills  the uterine cavity, has the tendency to reflux from the opening of the cervical canal due to the ovulation. (Franco et al., 1992).

Previous studies also showed good results with intratubal insemination (Lucena et al., 1989; Berger., 1987; Oei et al, 1992). However, problems arised in the catheterization of  the Fallopian tube and the possible complications associated with tubal cannulation of one of the tubes. With the FSP technique none of the above mentioned problems exist. In addition, the insemination fluid enters both Fallopian tubes, whereas tubal cannulation permits insemination of one tube only at a time. Other studies showed good results in pregnancy rates after Direct Peritoneal Insemination, rather  than those obtained by standard IUI. (Forrler et al.,1986 ; Seracchioli  et al., 1991; Crosignani et al., 1991). However this is an invasive method, not well tolerated by the patient.

   Finally, combined intra-uterine and direct intra-peritoneal insemination, has given favourable results with pregnancy rate per cycle of 30% ( Abyholm et al., 1992), but this is also an invasive method of insemination. With the use of the Double Nut Bivalve Speculum, most of the above mentioned problems are surmounted. In four cases with previous cervical cone excisions (2 knife cone, 2 Laser cone) and in 5 overweight patients the cervix had to be grasped and gently drawn to the outlet with a tenaculum.

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